Method for cancer immunotherapy

ABSTRACT

A novel compound of the formula (1): 
     
       
         
         
             
             
         
       
         
         
           
             wherein X is a tyrosine residue or a methionine residue; Y and Z each are a single bond or the like; R 1  is a hydrogen atom or the like; R 2  is a hydroxy group or the like; R 3  is a hydrogen atom, alkyl group, amino group or the like; R 4  is a hydrogen atom, alkyl group, carboxy group or the like; m is 1 or 2; and n is an integer of 0 to 2, with the proviso that when n is 0, R 3  is a hydrogen atom or an alkyl group, or a pharmaceutically acceptable salt thereof, and its use in cancer immunotherapy.

TECHNICAL FIELD

The present invention relates to cancer vaccine therapy, especially to anovel peptide compound useful as a medicament for cancer immunotherapy.In particular, the present invention relates to a derivative of a cancerantigen peptide derived from WT1, which has CTL induction activity invivo and is useful as cancer vaccine.

BACKGROUND ART

The cell mediated immunity, particularly a cytotoxic T cell(hereinafter, referred to as “CTL”) plays a significant role in the invivo rejection of cancer cells or virus-infected cells. CTLs recognize acomplex between an antigen peptide (“cancer antigen peptide”) derivedfrom a cancer antigen protein and an MHC (major histocompatibilitycomplex) class I antigen, which is referred to as “HLA antigen” in thecase of human, on a cancer cell, and attack and kill the cell.

A cancer antigen peptide which binds to MHC class 1 molecule isgenerally known to be a peptide having 8-12 amino acid residues producedby intracellular processing of the protein. Thus, in general, a peptidehaving 8-12 amino acid residues derived from a cancer antigen proteincan be a candidate for a cancer antigen peptide. When a glutamineresidue or a cysteine residue is present in the cancer antigen peptide,those amino acid residues are spontaneously oxidized in air atmospherein general. It is reported that such spontaneous oxidization decreasesbinding affinity of the peptide for MHC class I molecule and cognitiveresponse to the peptide by T cell receptor (see nonpatent literatures 1and 2).

The tumor suppressor gene WT1 of Wilms tumor (WT1 gene) has beenisolated from chromosome 11p13 as one of the causative genes of Wilmstumor based on analysis of the WAGR syndrome that occurs as acomplication of Wilms tumor, aniridia, urogenital abnormalities, mentalretardation and so forth, and the amino acid sequence of WT1 is publiclyknown (see nonpatent literature 3). The WT1 gene is expressed with highfrequency in human leukemia, and when leukemia cells are treated withWT1 antisense oligomers, the growth of the cells is inhibited. Thus, WT1gene is thought to act to promote the growth of leukemia cells.Moreover, WT1 gene is also highly expressed in solid cancers such asgastric cancer, colon cancer, lung cancer, breast cancer, embryonalcancer, skin cancer, bladder cancer, prostate cancer, uterine cancer,cervical cancer and ovarian cancer, and the WT1 gene has beendemonstrated to be a novel cancer antigen protein in leukemia and solidcancers (see nonpatent literatures 4 and 5). In addition, a cancerantigen peptide having a partial sequence of WT1 protein that is a wildtype cancer antigen peptide was identified (see patent literatures 1 and2).

Particularly, WT1₂₃₅₋₂₄₃ (Cys-Met-Thr-Trp-Asn-Gln-Met-Asn-Leu; SEQ IDNO: 1), that is a peptide spanning in positions 235 to 243 of the cancerantigen protein WT1, is a cancer antigen peptide having an activity toinduce CTLs in HLA-A24-restricted manner (see nonpatent literature 6 andpatent literature 1). The modified peptide(Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu; SEQ ID NO: 2, hereinafter it maybe referred to as WT1₂₃₅₋₂₄₃ (2M→Y)), in which the methionine residue atposition 2 of WT1₂₃₅₋₂₄₃ is replaced with tyrosine residue, has a higherbinding affinity for the HLA-A24 antigen than the wild type peptide (seepatent literature 3). The development of both wild type peptideWT1₂₃₅₋₂₄₃ and the modified peptide WT1₂₃₅₋₂₄₃ (2M→Y) as animmunotherapeutic agent is promising.

In addition, it is known that each of said wild type peptide and saidmodified peptide has a cysteine residue at the N-terminal site,oxidization of which in air atmosphere produces the dimer bound viadisulfide bond, and said dimer may also work as a cancer antigen peptide(see patent literature 4).

-   Patent literature 1: WO 00/06602-   Patent literature 2: WO 00/18795-   Patent literature 3: WO 02/079253-   Patent literature 4: WO 2004/063217-   Nonpatent literature 1: Immunity., 6:273, 1997-   Nonpatent literature 2: J. Immunol., 160:2099, 1998-   Nonpatent literature 3: Cell, 60:509, 1990-   Nonpatent literature 4: J. Immunol., 164:1873-80, 2000-   Nonpatent literature 5: J. Clin. Immunol., 20, 195-202, 2000-   Nonpatent literature 6: Clin. Cancer. Res. 8: 2626, 2002

DISCLOSURE OF INVENTION A Problem to be Solved by the Invention

A problem to be solved by the present invention is to provide a novelpeptide compound which has CTL induction activity in vivo and is usefulas cancer vaccine in cancer immunotherapy.

A Means for Solving the Problem

The inventors earnestly conducted various studies on modification of thecancer antigen peptides WT1₂₃₅₋₂₄₃ and WT1₂₃₅₋₂₄₃ (2M→Y) derived fromWT1 protein in order to produce a cancer antigen peptide having improvedphysical-chemical property, stability and bioactivity. In particular,they prepared the compounds modified from these peptides and examinedthe immunogenicity using HLA-A2402/Kb transgenic mice (see WO 02/47474,hereinafter they may be also referred to as HLA-A24 mice).

Consequently, they succeeded in preparing a peptide compound havingimproved physical-chemical property and stability by means of modifyingcysteine residue (Cys) at the N-terminal site of WT1₂₃₅₋₂₄₃ orWT1₂₃₅₋₂₄₃ (2M→Y) especially modifying the thiol group of the cysteineresidue at the N-terminal site. The peptide compound of the presentinvention has an improved immunogenicity and CTL induction activity. TheT cells specifically induced by the present peptide compound are usefulas a medicament for cancer immunotherapy due to their cross-reactionwith wild type peptide WT1₂₃₅₋₂₄₃ which is originally presented bycancer cells.

So far, the peptide compound in which the cysteine residue of WT1₂₃₅₋₂₄₃or WT1₂₃₅₋₂₄₃ (2M→Y) as the WT1 antigen peptide is modified was notknown to show the immunogenicity enough to work as a cancer antigen. Thepresent inventors firstly found that a peptide derivative prepared bycondensing the thiol group of cysteine residue at the N-terminal withthe thiol group of cysteine, glutathione or thioglycolic acid to form adisulfide bond, can be used as an effective cancer antigen.

The present invention was completed based on the finding as describedabove.

The present invention relates to

[1] a compound of the formula (1):

wherein X is a tyrosine residue or a methionine residue; each of Y and Zis independently selected from a single bond and a divalent peptidegroup consisting of 1-10 amino acid residues,R¹ is hydrogen or alkyl,R² is hydroxyl, amino, alkylamino or dialkylamino,R³ is hydrogen, alkyl, amino, alkylamino, dialkylamino or substituted orunsubstituted alkylcarbonylamino,R⁴ is hydrogen, alkyl, carboxy, carbamoyl, alkylcarbamoyl,dialkylcarbamoyl or a group of the formula (2):

-   -   wherein W is an amino acid residue,        m is 1 or 2, and        n is an integer of 0-2, with the proviso that when n is 0, R³ is        hydrogen or alkyl,        or a pharmaceutically acceptable salt thereof;        [2] the compound according to above [1], or a pharmaceutically        acceptable salt thereof,        wherein said substituted alkylcarbonylamino represented by        R³ is alkylcarbonylamino substituted by one or two substituent        group selected from the group consisting of carboxy, amino,        alkylamino and dialkylamino;        [3] the compound according to above [1] or [2], or a        pharmaceutically acceptable salt thereof,        R³ is hydrogen or a group of the formula (3):

-   -   wherein r is an integer of 1-3, and        R⁴ is carboxy or a group of the formula (2′):

-   -   wherein W′ is a glycine residue or β-alanine residue;        [4] the compound according to above [3], or a pharmaceutically        acceptable salt thereof,        wherein R³ is a group of the formula (3′):

andR⁴ is carboxymethylcarbamoyl;[5] the compound according to above [3], or a pharmaceuticallyacceptable salt thereof,wherein R³ is hydrogen and R⁴ is carboxy;[6] the compound of the formula (1′):

-   -   wherein X′ is a tyrosine residue or a methionine residue,    -   R^(1′) is hydrogen or alkyl,    -   R^(2′) is hydroxyl, amino, alkylamino or dialkylamino,    -   R^(3′) is amino, alkylamino, dialkylamino or substituted or        unsubstituted alkylcarbonylamino, and    -   R^(4′) is carboxy, carbamoyl, alkylcarbamoyl or        dialkylcarbamoyl,        or a pharmaceutically acceptable salt thereof;        [7] an antibody which specifically binds to the compound        according to any one of above [1]-[6] or a pharmaceutically        acceptable salt thereof;        [8] an antigen-presenting cell which presents a complex of the        compound according to any one of above [1]-[6] or a        pharmaceutically acceptable salt thereof and HLA-A24 antigen;        [9] a CTL induced by the compound according to any one of above        [1]-[6] or a pharmaceutically acceptable salt thereof;        [10] the CTL according to above [9], which recognizes a complex        of the compound according to any one of above [1]-[6] or a        pharmaceutically acceptable salt thereof and HLA-A24 antigen;        [11] the CTL according to above [9], which recognizes a complex        of the peptide of SEQ ID No:1 and HLA-A24 antigen;        [12] a pharmaceutical composition comprising the compound        according to any one of above [1]-[6] or a pharmaceutically        acceptable salt thereof, the antigen-presenting cell according        to above [8] or the CTL according to any one of above [9]-[11],        together with a pharmaceutically acceptable carrier;        [13] the pharmaceutical composition according to above [12],        which is used as a cancer vaccine;        [14] a use of the compound according to any one of above [1]-[6]        or a pharmaceutically acceptable salt thereof, the        antigen-presenting cell according to above [8] or the CTL        according to any one of above [9]-[11] for preparing a cancer        vaccine;        [15] a medicament for cancer immunotherapy comprising the        compound according to any one of above [1]-[6] or a        pharmaceutically acceptable salt thereof, the antigen-presenting        cell according to above [8] or the CTL according to any one of        above [9]-[11] as an active ingredient; or        [16] a method for the treatment or prevention of cancer, which        comprises administrating a therapeutically or prophylactically        effective amount of the compound according to any one of above        [1]-[6] or a pharmaceutically acceptable salt thereof, the        antigen-presenting cell according to above [8] or the CTL        according to any one of above [9]-[11] to a patient who is        positive for HLA-A24 and positive for WT1 in need of the        treatment or prevention of the cancer.

The Effect Produced by the Present Invention

A novel peptide compound useful as a medicament for cancerimmunotherapy, for example, a cancer antigen derived from WT1 which hasCTL induction activity in vivo and is useful as cancer vaccine isprovided by the present invention. The peptide of the present invention,which is prepared by modification of mercapto group of cysteine residuelocated at N-terminal of WT1₂₃₅₋₂₄₃ or WT1₂₃₅₋₂₄₃ (2M→Y) withmaintaining the activity as a cancer antigen peptide, has improvedphysical-chemical property and stability and thus, it can be widely usedfor treatment or research. In particular, the novel peptide of thepresent invention has advantages such as convenience in handling withouttaking care of decrease in activity due to in vitro treatment,exhibition of stable therapeutic effect and so on.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing cytotoxic activity (Specific Lysis) induced bythe peptide compound of Example 1 using three mice individually (blackbar in the figure). In the figure, white bar shows the results obtainedusing cells not pulsed with any peptide (The same is applicable to thefollowing figures).

FIG. 2 is a graph showing cytotoxic activity (Specific Lysis) induced bythe peptide compound of Example 2 using three mice individually.

FIG. 3 is a graph showing cytotoxic activity (Specific Lysis) induced bythe peptide compound of Example 3 using three mice individually.

FIG. 4 is a graph showing cytotoxic activity (Specific Lysis) induced bythe peptide compound of Example 4 using three mice individually.

FIG. 5 is a graph showing cytotoxic activity (Specific Lysis) induced bythe peptide compound of Example 5 using three mice individually.

FIG. 6 is a graph showing cytotoxic activity (Specific Lysis) induced bythe peptide compound of Example 6 using three mice individually.

FIG. 7 is a graph showing dose-dependent cytotoxic activity (SpecificLysis) induced by the peptide compound of Example 1. The X axis showsdose per one individual mouse (600 μg, 200 μg, 60 μg and 20 μg), and Yaxis shows cytotoxic activity (Specific Lysis). Each dose wasadministrated to three mice respectively and the results are shown asmean of the cytotoxic activities and the standard deviation (S.D.).

FIG. 8 is a graph showing reactivity of the peptide-specific T cellprepared by immunization of mice with the peptide compound of Example 1to cells pulsed with various peptides. In the figure, the hatched barshows the result obtained using cells pulsed with the wild type peptide(WT1₂₃₅₋₂₄₃), the black bar shows the result obtained using cells pulsedwith the peptide compound of Example 1 and the white bar shows theresult obtained using cells pulsed with a peptide derived fromInfluenza, respectively. In addition, in the figure, the vertical axisshows spots.

FIG. 9 is a graph showing reactivity of the peptide-specific T cellderived from human PBMC by stimulation with the peptide of Example 1 tocells pulsed with various peptides. In the figure, “wild type peptide”shows the result obtained using cells pulsed with the wild type peptide(WT1₂₃₅₋₂₄₃), the “modified peptide” shows the result obtained usingcells pulsed with the modified peptide (WT1₂₃₅₋₂₄₃ (2M→Y)), the “peptideof Example 1” shows the results obtained using cells pulsed with thepeptide compound of Example 1 and “without peptide pulse” shows theresult obtained using cells not pulsed with any peptide, respectively.In addition, in the figure, the vertical axis shows the amount ofproduced interferon-γ.

BEST MODE FOR CARRYING OUT THE INVENTION

As used in the specification and the drawings of the presentapplication, the following abbreviations are used for each amino acidresidue.

Ala: alanine residueArg: arginine residueAsn: asparagine residueAsp: aspartic acid residueCys: cysteine residueGln: glutamine residueGlu: glutamic acid residueGly: glycine residueHis: histidine residueIle: isoleucine residueLeu: leucine residueLys: lysine residueMet: methionine residuePhe: phenylalanine residuePro: proline residueSer: serine residueThr: threonine residueTrp: tryptophan residueTyr: tyrosine residueVal: valine residue

As used in the specification, “amino acid residue” includes natural ornonnatural α-amino acid residue, β-amino acid residue, γ-amino acidresidue and δ-amino acid residue. For example, “amino acid residue”includes a natural α-amino acid (for example, Ala, Arg, Asn, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr orVal), ornithine residue, homoserine residue, homocysteine residue,β-alanine, γ-aminobutanoic acid or δ-aminopentanoic acid.

The above described amino acid can be either L-enantiomer orD-enantiomer when it is an optical isomer. L-enantiomer is morepreferable.

In the specification, the amino acid sequence of the peptide compound isdescribed according to the conventional style where the N-terminal aminoacid residue is located on the left side and the C-terminal amino acidresidue is located on the right side.

(1) Peptide Compound

In a first aspect, the present invention relates to the compound of theabove described formula (1) or a pharmaceutically acceptable saltthereof.

In the formula (1), preferably, X represents a tyrosine residue (Tyr).

In the formula (1), “the divalent peptide groups consisting of 1-10amino acid residues” represented by Y and Z include the same ordifferent divalent peptide group consisting of 1-10 amino acid residueswithout any limitation of the amino acid sequence. For example, saiddivalent peptide group consisting of 1-10 amino acid includes an aminoacid sequence comprised in the amino acid sequence of human WT1 (Cell,60:509, 1990, GenBank Acc. No. A38080). For example, Y may represent adivalent peptide group consisting of ten amino acid residues of 225 to234 of human WT1, which is described as follows:Asn-Leu-Tyr-Gln-Met-Thr-Ser-Gln-Leu-Glu (SEQ ID No: 3) or a divalentpeptide group having a fragment of SEQ ID NO: 3, in which 1-9 amino acidresidues at N-terminal are deleted. In addition, for example, Z mayrepresent a divalent peptide group consisting of ten amino acid residuesof 244 to 253 of human WT1, which is described as follows:Gly-Ala-Thr-Leu-Lys-Gly-Val-Ala-Ala-Gly (SEQ ID No: 4) or a divalentpeptide group having a fragment of SEQ ID NO: 4, in which 1-9 amino acidresidues at C-terminal are deleted. Preferably, each of Y and Z mayrepresent a single bond.

In the specification, the alkyl group includes, for example, a linear orbranched alkyl group having 1-6 carbon atoms. For example, the alkylgroup includes methyl, ethyl, propyl, 1-methylethyl, butyl,1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl and the like.

In the specification, the alkylamino group includes, for example, alinear or branched alkylamino group having 1-6 carbon atoms. Forexample, the alkylamino group includes methylamino, ethylamino,propylamino, 1-methylethylamino, butylamino, 1-methylpropylamino,2-methylpropylamino, 1,1-dimethylethylamino, pentylamino and the like.

In the specification, the dialkylamino group includes, for example, anamino group substituted by two same or different and linear or branchedalkyl groups having 1-6 carbon atoms. For example, the dialkylaminogroup includes dimethylamino, ethylmethylamino, diethylamino,dipropylamino, methylpropylamino, butylmethylamino, methylpentylaminoand the like.

In the specification, the “alkyl” of the alkylcarbonylamino groupincludes the alkyl group as described above. The “alkyl” of thealkylcarbamoyl group includes the same alkyl as that of alkylamino groupas described above. The “alkyl” of the dialkylcarbamoyl group includesthe same alkyl as that of dialkylamino group as described above, whereintwo “alkyl” may be same or different.

Each of R¹ and R² preferably represents a hydrogen atom.

When R³ represents a substituted alkylcarbonylamino group, thesubstituted group of alkylcarbonylamino group includes, for example,carboxy, hydroxyl, amino, alkylamino and dialkylamino, wherein saidalkylcarbonylamino group may be substituted by same or different 1-4,preferably 1 or 2 substituted groups.

The amino acid residue represented by W in the formula (2) may includeglycine residue (Gly), preferably.

The peptide compound of the present invention includes, for example,compounds of the following formulae (4)-(9):

The peptide compounds of the present invention may be prepared accordingto the method described in the Examples of the specification or a methodusually used in peptide synthesis. Examples of such preparations arethose as described in the literatures including “Peptide Synthesis”,Interscience, New York, 1966; “The Proteins”, Vol. 2, Academic PressInc., New York, 1976; “Pepuchido-Gosei”, Maruzen Co. Ltd., 1975;“Pepuchido-Gosei-no-Kiso-to-Jikkenn”, Maruzen Co. Ltd., 1985; and“Iyakuhin-no-Kaihatu, Zoku, vol. 14, Peputido-Gosei”, Hirokawa Shoten,1991. Example of the method for preparing the peptide of the presentinvention includes a method for preparing the peptide according to Fmocmethod or Boc method by means of solid-phase synthesizer or a method forpreparing the peptide by means of sequential condensation of Boc-aminoacid or Z-amino acid according to the liquid-phase synthesis method,wherein Fmoc represents 9-fluorenylmethyloxycarbonyl group, Bocrepresents tert-butoxycarbonyl group and Z represents benzyloxycarbonylgroup, respectively.

A functional group such as amino, carboxyl and mercapto group of theintermediate compound in the synthesis of the compound of the presentinvention may be protected by a suitable protective group, and theprotected compound may be deprotected using a conventional technique ofprotection/deprotection, if required. A suitable protective group and amethod for the protection and the deprotection are described forexample, in Protective Groups in Organic Synthesis 2nd Edition (JohnWiley & Sons, Inc.; 1990), in detail.

In particular, an example of the method for preparing the compound ofthe present invention is a method as described in the following reactionformulae:

-   -   wherein X, Y, Z, R¹, R², R³, R⁴, m and n are same as described        above, respectively. Each of R and R′ independently represents a        hydrogen atom or a protective group for mercapto group.    -   The example of said protective group for mercapto group        includes, for example, acetoamidomethyl or trityl.

The compound of the formula (1) may be prepared by oxidizing thecompound of the formula (1-1) and the compound of the formula (1-2) inan inert solvent.

The oxidization may be conducted by a conventional method normallyapplied in the peptide synthesis to form a disulfide bond. For example,a disulfide bond may be formed by mixing two intermediates havingmercapto group in a suitable solvent and oxidizing them. A conventionalmethod for oxidization such as air oxidation and iodine oxidation may beused. The solvent may be water, acetic acid, methanol, chloroform, DMFor DMSO or a mixture thereof. The oxidation reaction sometimes gives amixture of the symmetrical and the asymmetrical disulfide compounds. Thedesired asymmetrical disulfide compound may be prepared by purificationof the mixture such as purification using various kinds ofchromatography, purification according to recrystallization method andthe like.

Alternatively, an intermediate having an activated mercapto group ismixed with another intermediate having a mercapto group to produce aselective disulfide bonding. Examples of the intermediate having anactivated mercapto group include, for example, a compound having amercapto group bound to Npys group (3-nitro-2-pyridine sulfenyl group).

Alternatively, after one of the intermediates having a mercapto group ismixed with, for example, 2,2′-dithiobis(5-nitropyridine) to activate themercapto group, the other intermediate is added to the resulting mixtureto form a selective disulfide bonding (Tetrahedron Letters. Vol. 37. No.9, pp. 1347-1350).

The compound of the formula (1-1) may be prepared according to aliquid-phase or a solid-phase peptide synthesis method which is wellknown by those skilled in the art.

In addition, in the case where the compound of the formula (1-1) is a Nterminally-alkylated compound, N-alkyl amino acid or N,N-dialkyl aminoacid which may be, if required, protected by a protective group may beused as a N-terminal amino acid. The N-alkyl amino acid or N,N-dialkylamino acid may be commercially available or prepared according to amethod well known by those skilled in the art, in which, for example, anamino acid or protected amino acid of starting material is reacted withan alkylhalide in the presence of base. For example, N-terminal aminogroup may be appropriately alkylated by reacting amino acid which isprotected by t-butoxycarbonyl group with alkylhalide in the presence ofbase such as sodium hydride as illustrated in the following [Reactionformula 2].

-   -   wherein each of X, Z, R¹, R², m and R is the same as described        above, Hal represents a bromine atom or an iodine atom, and Prot        represents a protective group.

Further, in the case where the compound is a C terminally-amidated oralkylamidated compound, an amidated or alkylamidated amino acid may beused as a C-terminal amino acid residue of the starting material.

The compound of the present invention or intermediates in the synthesisthereof may be purified according to the method well known by thoseskilled in the art. For example, the purification may be conducted bymeans of various kinds of chromatography (e.g. silica-gel columnchromatography, ion-exchange column chromatography, gel filtrationchromatography or reversed phase chromatography) or recrystallization.The solvent for the recrystallization may be, for example, alcohols suchas methanol, ethanol and 2-propanol, ethers such as diethylether, esterssuch as ethyl acetate, aromatic hydrocarbons such as benzene andtoluene, ketones such as acetone, hydrocarbons such as hexane, aproticsolvents such as dimethylformamide and acetonitrile, water or mixedsolvents thereof. Other method used for the purification may be themethod as described in volume 1 of Jikkenkagakukoza (edited by ChemicalSociety of Japan, Maruzen).

In the case where the compound of the present invention has one or moreasymmetric centers, the material (amino acid) having the asymmetriccenter may be used for preparing it according to the conventionalmethod. In addition, the optical resolution may be carried out in theappropriate step of the production process to improve the optical purityof the compound of the present invention. For example, the opticalresolution may be carried out according to the diastereomer method inwhich the compound or the intermediate of the present invention is mixedwith optically active acid (e.g. monocarboxylic acid such as mandelicacid, N-benzyloxyalanine and lactic acid, dicarboxylic acid such astartaric acid, o-diisopropylidene tartaric acid and malic acid orsulfonic acid such as camphorsulfonic acid and bromocamphorsulfonicacid) in an inactive solvent (e.g. alcohols solvent such as methanol,ethanol and 2-propanol, ethers such as diethylether, esters solvent suchas ethyl acetate, hydrocarbons solvent such as toluene, aprotic solventssuch as acetonitrile or mixed solvents thereof) to prepare the saltform. In the case where the compound or the intermediate of the presentinvention has an acid functional group such as carboxy group, theoptical resolution may be carried out by forming the salt with opticallyactive amine (e.g. organic amine such as α-phenethylamine, quinine,quinidine, cinchonidine, cinchonine and strychnine).

The reaction for forming the salt may be conducted at a temperatureranging from a room temperature to boiling point of the solvent. For thesake of improving the optical purity, it is preferable that thetemperature is once increased up to about the boiling point of thesolvent. The yield may be improved, if required by cooling the mixture,when the precipitated salt is recovered by filtration.

The optically active acid or amine is appropriately used in the amountof about 0.5 to about 2.0 equivalents to the substrate, preferably inthe amount of about 1 equivalent to the substrate. If required, thecrystal may be recrystallized in an inactive solvent (e.g. alcohols suchas methanol, ethanol and 2-propanol, ethers such as diethylether, esterssuch as ethyl acetate, hydrocarbons such as toluene, aprotic solventssuch as acetonitrile or mixed solvents thereof) to obtain the highlypurified optically active salt. In addition, if required, the opticallyresolved salt may be treated with acid or base according to theconventional method in order to obtain the compound of free form.

A pharmaceutically acceptable salt includes an acid addition salt and abase addition salt. For example, the acid addition salt includes a saltwith an inorganic acid such as hydrochloride, hydrobromide, sulfate,hydroiodide, nitrate and phosphate, and a salt with an organic acid suchas citrate, oxalate, acetate, formate, propionate, benzoate,trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonateand paratoluenesulfonate. The base addition salt includes a salt with aninorganic base such as sodium salt, potassium salt, calcium salt,magnesium salt and ammonium salt, a salt with an organic base such astriethylammonium salt, triethanol ammonium salt, pyridinium salt anddiisopropylammonium salt, and further a salt of amino acid such as basicor acidic amino acid including arginine, asparagine acid and glutamicacid.

In addition, the present invention comprises the solvate of the peptidecompound represented by the formula (1) or a pharmaceutically acceptablesalt thereof including hydrate or ethanol solvate. Further, the presentinvention comprises any possible stereoisomers including anydiastereomers and enantiomers of the compound represented by formula(1), and any crystal forms thereof.

In the course of preparing a peptide compound including the steps ofcondensing an optically active α-amino acid, removing various kinds ofprotecting group or releasing the peptide from resin, by-productsincluding a peptide with amino acid deletion, a peptide degraded byhydrolysis, oxidization or the like, and a peptide having a epimerizedamino acid are usually produced. At laboratory scale, a combination ofvarious chromatographies (e.g. silica-gel column chromatography,ion-exchange column chromatography, gel filtration or reversed phasechromatography) may be used for removing those impurities so as toobtain a highly purified peptide compound. However, it is not easy toobtain the highly purified peptide compound at industrial scale for thesake of providing it as a medicine.

The peptide compound of the present invention has a physical-chemicalproperty which enables the large scale production of the bulkpharmaceuticals. In particular, the peptide compound of the presentinvention has a property including high solubility, high stability inthe solution or a tendency not to turn into a gel when it is condensedso that the highly purified peptide compound may be easily prepared as abulk pharmaceutical even in the large scale by means of the purificationusing column chromatography such as reversed phase chromatography.

The peptide compound of the present invention is useful for an activeingredient comprised in CTL inducer or cancer vaccine for cancerimmunotherapy. The peptide compound of the present invention has a highimmunogenicity and high CTL induction activity as shown in Examples ofthe present specification. The CTL induced by the peptide compound ofthe present invention can surprisingly recognize a wild type peptide ofWT1 originally carried by cancer cells. Thus, the peptide compound ofthe present invention is useful for a medicament for the treatment orprevention (including prevention of a recurrence) of cancer expressingWT1 gene such as gastric cancer, colon cancer, lung cancer, breastcancer, embryonal cancer, skin cancer, bladder cancer, prostate cancer,uterine cancer, cervical cancer, and ovarian cancer.

(2) Antibodies of the Present Invention

In a second aspect, the present invention relates to antibodies whichspecifically bind to a peptide represented by the formula (1) orpharmaceutically acceptable salt thereof (hereinafter they may bereferred to as “antibodies of the invention). The antibodies of theinvention are not limited to a specific antibody, and may be apolyclonal antibody or a monoclonal antibody prepared using a peptide ofthe present invention as an immunogen.

The antibodies of the present invention are not limited to a specificantibody as long as they specifically bind to the peptide compounds ofthe invention, and specific examples include an antibody thatspecifically binds to a peptide represented by any one of the formulae(4)-(9) as described above.

A method for preparing the antibodies have been already well known, andthe antibodies of the present invention may be prepared according toconventional methods (Current protocols in Molecular Biology edit.Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12 to11.13, Antibodies; A Laboratory Manual, Lane, H, D. et al. ed., ColdSpring Harber Laboratory Press Publisher, New York 1989).

Specifically, the antibodies may be prepared using the peptide compoundsof the present invention (for example, a compound represented by any oneof the formulae (4)-(9)) as an immunogen to immunize a non-human animalsuch as a rabbit, followed by obtaining the antibodies from the serum ofthe immunized animal in a conventional manner. On the other hand,monoclonal antibodies may be prepared by immunizing a non-human animalsuch as a mouse with the compound of the present invention, for example,a compound represented by any one of the formulae (4)-(9), and preparinghybridoma from the splenocytes obtained from the animal and myelomacells by means of cell fusion, followed by obtaining the antibodies fromthe hybridoma (Current protocols in Molecular Biology edit. Ausubel etal. (1987) Publish. John Wiley and Sons. Section 11.4 to 11.11).

The antibodies directed to the peptide compounds of the invention may beprepared in a manner that the immunological reaction is enhanced usingdiverse adjuvants suitable for the host. Examples of the adjuvantsinclude Freund's adjuvant, mineral gels such as aluminium hydroxide,surfactants such as lysolecithin, Pluronic polyol, polyanions, peptides,oil emulsions, Keyhole limpet Hemocyanin, and dinitrophenol, and humanadjuvants such as BCG (Bacille Calmette Guerin) andCorynebacterium-parvum.

As described above, the antibodies that recognize the compound of thepresent invention, as well as the antibodies that neutralize theactivity of the compound may be readily prepared by immunizingappropriately an animal with the compounds of the present invention in aconventional manner. Such antibodies may be used in affinitychromatography, immunological diagnosis, and the like. Immunologicaldiagnosis may be appropriately selected from immunoblotting,radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), afluorescent or luminescent assay, and the like. The immunologicaldiagnosis is useful to diagnose cancers expressing the WT1 gene, such asgastric cancer, colon cancer, lung cancer, breast cancer, embryonalcancer, skin cancer, bladder cancer, prostate cancer, uterine cancer,cervical cancer, and ovarian cancer.

(3) Antigen-Presenting Cells of the Present Invention

In a third aspect, the present invention relates to antigen-presentingcells on which a complex between a compound of the present invention andan HLA-A24 antigen is presented.

Examples described hereinafter demonstrate that the administration ofthe compounds of the present invention induces CTLs. That is,antigen-presenting cells on which a complex between the compound of thepresent invention and an HLA-A24 antigen is presented, are generated inthe peripheral blood mononuclear cells, and then CTLs which specificallyrecognize the cells presenting such a complex are induced. Thoseantigen-presenting cells on which a complex between an HLA-A24 antigenand the compound of the present invention is presented, are useful incell therapy (DC therapy) as described hereinafter.

Antigen-presenting cells of the present invention are not limited to aspecific cell as long as they presents on their surfaces a complexbetween the compound of the present invention and an HLA-A24 antigen.They specifically include, for example, antigen-presenting cells ofdendritic cells on which a complex between the compound represented byany one of the formulae (4)-(9) and an HLA-A24 antigen is presented.

Antigen-presenting cells used in cell therapy (DC therapy) may beprepared by isolating cells having an antigen-presenting ability from acancer patient and pulsing the resulting cells ex vivo with the compoundof the present invention so that the cells present a complex between acompound of the present invention and an HLA-A24 antigen on their cellsurface. In this context, the “cell having an antigen-presentingability” is not limited to a specific cell as long as it is a cellexpressing on the surface an HLA-A24 antigen that has an ability topresent the compound of the present invention, and dendritic cells,which is believed to have an especially high antigen-presenting ability,are preferably exemplified.

Antigen-presenting cells of the present invention may be prepared, forexample, by isolating cells having an antigen-presenting ability from acancer patient, pulsing the cells ex vivo with the compound of theinvention (e.g. the compound of any one of the formulae (4)-(9)), andpreparing a complex between an HLA-A24 antigen and the compound of thepresent invention (Cancer Immunol. Immunother., 46: 82, 1998, J.Immunol., 158: p 1796, 1997, Cancer Res., 59: p 1184, 1999). Whendendritic cells are used, antigen-presenting cells of the presentinvention may be prepared, for example, by isolating lymphocytes fromperipheral bloods of a cancer patient using Ficoll method, removing thenon-adherent cells, incubating the adherent cells in the presence ofGM-CSF and IL-4 to induce dendritic cells, and incubating and pulsingthe resultant dendritic cells with the compound of the presentinvention.

The antigen-presenting cells thus prepared as described above are usefulas an active ingredient comprised in a CTL inducer or a cancer vaccinefor cell therapy (DC therapy) as described hereinafter.

(4) CTLs of the Present Invention

The peptide compounds of the present invention are derived from humanWT1 and have a CTL induction activity (immunogenicity) inHLA-A24-restricted manner. In a fourth aspect, the present inventionrelates to CTL induced by a peptide compound of the present invention.

Examples described hereinafter demonstrate that the administration ofthe peptide compounds of the present invention induces CTLs. That is,antigen-presenting cells, on which a complex between a compound of thepresent invention and an HLA-A24 antigen is presented, are generated inthe peripheral blood mononuclear cells, and then CTLs which specificallyrecognize the cells presenting such a complex are induced. Those CTLsinduced by the peptide compound of the present invention are useful inadoptive immunotherapy as described hereinafter.

CTLs of the present invention are not limited to a specific CTL as longas they are induced by the peptide compound of the present invention,and particularly include CTLs recognizing a complex between the compoundrepresented by any one of the formulae (4)-(9) and an HLA-A24 antigenand CTLs recognizing a complex between the wild type peptide(WT1₂₃₅₋₂₄₃: SEQ ID No:1) and an HLA-A24 antigen.

CTLs used in adoptive immunotherapy may be prepared by, for example,isolating peripheral lymphocytes from a patient and stimulating theresulting peripheral lymphocytes in vitro with the compound of thepresent invention (e.g. the compound represented by any one of theformulae (4)-(9)) (Journal of Experimental Medicine 1999, 190: 1669).

The CTLs of the present invention prepared as described above are usefulas an active ingredient comprised in a cancer vaccine for an adoptiveimmunotherapy.

Pharmaceutical Compositions Usable as Cancer Vaccines

Compounds of the present invention, antigen-presenting cells of thepresent invention and CTLs of the present invention as described above(1)-(4) may be used as an active ingredient comprised in CTL inducer,that is to say a cancer vaccine, when formulated into a form asappropriate for those respective substances, which are illustratedbelow.

1) Cancer Vaccines Comprising a Compound of the Present Invention or aPharmaceutical Salt Thereof as an Active Ingredient

The compound of the present invention has CTL induction activity. CTLsinduced by the compound of the present invention can destroy cancers viatheir cytotoxic activity and the lymphokine productions. Thus, thecompounds of the present invention can be used as an active ingredientcomprised in a cancer vaccine for treatment or prevention of cancers. Inthe embodiment, the invention provides a cancer vaccine which comprisesas an effective ingredient the compounds of the invention (apharmaceutical composition usable as cancer vaccines). When the cancervaccine of the invention is administered to a cancer patient positivefor HLA-A24 and positive for WT1, the compound (e.g. the compoundrepresented by any one of the formulae (4)-(9)) is presented on anHLA-A24 antigen of antigen-presenting cells, and then CTLs specific forthe presented complex comprising the HLA-A24 antigen efficientlyproliferate, and destroy cancer cells. In this way, treatment orprevention of cancers is achieved. The cancer vaccines of the inventioncan be used to treat or prevent cancers which involve the elevatedexpression level of the WT1 gene, including blood cancers such asleukemia, myelodysplastic syndrome, multiple myeloma and malignantlymphoma, and solid cancers such as gastric cancer, colon cancer, lungcancer, breast cancer, embryonal cancer, hepatic cancer, skin cancer,bladder cancer, prostate cancer, uterine cancer, cervical cancer, andovarian cancer. In this connection, as another embodiment, the inventionprovides a method for the treatment or prevention of cancer, whichcomprises administering an effective amount of the cancer vaccine of thepresent invention to a cancer patient who is positive for an HLA-A24,and positive for WT1.

The cancer vaccine comprising a compound of the present invention as anactive ingredient may comprise either a single cancer antigen peptide,that is to say, CTL epitope (e.g. a compound represented by any one ofthe formulae (4)-(9)) as an active ingredient, or an epitope peptideconnected to another cancer antigen peptide (a CTL epitope) or a helperepitope as an active ingredient. Recently, it has been demonstrated thatan epitope peptide connected to a plurality of CTL epitopes (antigenpeptides) has an activity to induce CTLs effectively in vivo. Forexample, Journal of Immunology 1998, 161: 3186-3194 describes that anabout 30-mer epitope peptide to which HLA-A2, -A3, -A11, B53-restrictedCTL epitopes (antigen peptides) derived from the cancer antigen proteinPSA are linearly-connected induced CTLs specific for the relevant CTLepitope in vivo. Also, it has been demonstrated that an epitope peptidein which a CTL epitope and a helper epitope are linearly-connectedeffectively induced CTLs. When a peptide of the invention in the form ofsuch epitope peptides is administered, the peptide is introduced intoantigen-presenting cells, and then subjected to intracellulardegradation to generate respective antigen peptides, which bind an HLAantigen to form complexes. The complexes are presented compactly on thecell surface of the antigen-presenting cells, and then CTLs specific forthe complexes efficiently proliferate in vivo, and destroy cancer cells.In this way, the treatment or prevention of cancers is achieved.

Cancer vaccines comprising the peptide of the present invention as anactive ingredient may be also administered together with apharmaceutically acceptable carrier such as a suitable adjuvant, or in aparticulate dosage form in order to effectively establish the cellularimmunity. For such purpose, those adjuvants described in the literature(Clin. Microbiol. Rev., 7:277-289, 1994) are applicable, andspecifically include bacterium-derived components, GM-CSF, cytokinessuch as Interleukin-2, Interleukin-7, Interleukin-12 and the like,plant-derived components, marine organism-derived component, mineralgels such as aluminium hydroxide, surfactants such as lysolecithin andPluronic polyol, polyanions, peptides, and oil emulsions (emulsionformulations). The bacterium-derived components include lipid A,monophosphoryl lipid A which is a derivative of lipid A, killedbacterium (e.g. Mycobacterium such as BCG), protein or polynucleotidederived from bacterium, Freund's Incomplete Adjuvant, Freund's CompleteAdjuvant, cell wall component (e.g. BCG-CWS), trehalose-dimycolate (TDM)and the like. Also, liposomal formulations, particulate formulations inwhich the ingredient is bound to beads having a diameter of several μm,or formulations in which the ingredient is attached to lipids are alsopossible.

Administration may be achieved by, for example, intradermal,subcutaneous, intramuscular or intravenous injection. Although the doseof the compound of the present invention in the formulations may varydepending on the disease to be treated, the age and the weight of thepatient, and the like, it is typical to administer 0.0001 mg to 1000 mg,preferably 0.001 mg to 1000 mg, more preferably 0.1 mg to 10 mg of acompound of the invention every several days to every several months.

2) Cancer Vaccines Comprising the Antigen-Presenting Cell of the PresentInvention as an Active Ingredient

The present invention provides a cancer vaccine which comprises theantigen-presenting cell of the present invention as an activeingredient.

Recently, cell therapy (DC therapy) has been reported whereinlymphocytes are isolated from the peripheral bloods of a cancer patient,and the dendritic cells induced from the lymphocytes are pulsed in vitrowith an antigen peptide or the like to prepare antigen-presenting cells,which are then returned into the patient via a subcutaneous injection orthe like (Cancer Immunol. Immunother., 46: 82, 1998, J. Immunol., 158: p1796, 1997, Cancer Res., 59: p 1184, 1999, Cancer Res., 56: p 5672,1996, J. Immunol., 161: p 5607, 1998, J. Exp. Med., 184: p 465, 1996).Thus, the antigen-presenting cell of the present invention can be usedas an active ingredient in a cancer vaccine in cell therapy.

The cancer vaccine which comprises the antigen-presenting cells of theinvention as an active ingredient preferably contains physiologicalsaline, phosphate buffered saline (PBS), medium, or the like to stablymaintain the antigen-presenting cells. It may be administered, forexample, intravenously, subcutaneously, or intradermally. The dose isexemplified by those described in the aforementioned literatures.

By reintroducing the cancer vaccine into the body of the patient,specific CTLs are efficiently induced in patients positive for HLA-A24,and positive for WT1 so as to achieve the treatment or the prevention ofthe cancers. The cancer vaccine which comprises the antigen-presentingcells of the invention as an active ingredient can be used to treat orprevent cancers wherein the level of the WT1 gene expression iselevated, including blood cancers such as leukemia, myelodysplasticsyndrome, multiple myeloma and malignant lymphoma, and solid cancerssuch as gastric cancer, colon cancer, lung cancer, breast cancer,embryonal cancer, hepatic cancer, skin cancer, bladder cancer, prostatecancer, uterine cancer, cervical cancer, and ovarian cancer.

3) Cancer Vaccines Comprising the CTL of the Present Invention as anActive Ingredient

The present invention provides a cancer vaccine which comprises as anactive ingredient the CTL of the invention (a pharmaceutical compositionusable as cancer vaccines). The CTL of the present invention are usefulin adoptive immunotherapy as described hereinafter.

For melanomas, it has been observed that an adoptive immunotherapyachieves a therapeutic effect, wherein tumor-infiltrating T cellsisolated from the patient himself/herself are cultured ex vivo in largequantities, and then returned into the patient (J. Natl. Cancer. Inst.,86:1159, 1994). Likewise, in mouse melanoma, suppression of metastasishas been observed by in vitro stimulation of splenocytes with cancerantigen peptide TRP-2, thereby proliferating CTLs specific for thecancer antigen peptide, and administering said CTLs into amelanoma-grafted mouse (J. Exp. Med., 185:453, 1997). This resulted fromin vitro proliferation of CTLs that specifically recognize the complexbetween an HLA antigen and the cancer antigen peptide onantigen-presenting cells. Accordingly, a method for treating cancers,which comprises stimulating in vitro peripheral blood lymphocytes from apatient using the compound of the present invention to proliferatetumor-specific CTLs in vitro, and subsequently returning the CTLs intothe patient, is believed to be useful. Thus, the CTLs of the inventionmay be used as an active ingredient comprised in cancer vaccine used inadoptive immunotherapy.

A cancer vaccine which comprises the CTLs of the present invention as anactive ingredient preferably contains physiological saline, phosphatebuffered saline (PBS), medium, or the like to stably maintain the CTLs.It may be administered, for example, intravenously, subcutaneously, orintradermally. The dose is exemplified by those described in theaforementioned literatures.

By reintroducing the cancer vaccine into the body of the patient,cytotoxic effect of CTLs on cancer cells is enhanced in the patientpositive for HLA-A24 and positive for WT1, and destroys cancer cells, soas to achieve the treatment of the cancers. The cancer vaccine whichcomprises the CTL of the present invention as an active ingredient canbe used to treat or prevent cancers which involve the elevated level ofthe WT1 gene expression. Examples of cancers include blood cancers suchas leukemia, myelodysplastic syndrome, multiple myeloma and malignantlymphoma, and solid cancers such as gastric cancer, colon cancer, lungcancer, breast cancer, embryonal cancer, hepatic cancer, skin cancer,bladder cancer, prostate cancer, uterine cancer, cervical cancer, andovarian cancer.

The present invention is further illustrated by the following examples,but is not limited by those examples in any respect.

In the following examples, the compound of Example 2, the compound ofExample 4 and the compound of Example 6 are derivatives of WT1₂₃₅₋₂₄₃(SEQ ID No:1), and correspond to the derivative of WT1₂₃₅₋₂₄₃ modifiedwith cystine, glutathione or thioglycolic acid, respectively. Inaddition, the compound of Example 1, the compound of Example 3 and thecompound of Example 5 are derivatives of WT1₂₃₅₋₂₄₃ (2M→Y) (SEQ IDNo:2), and correspond to the derivative of WT1₂₃₅₋₂₄₃ (2M→Y) modifiedwith cystine, glutathione or thioglycolic acid, respectively.

EXAMPLE

The abbreviations used in the Examples are as follows:

Boc: t-butoxycarbonylNpys: 3-nitro-2-pyridine sulfenylt-Bu; t-butylTrt: triphenylmethylFmoc: 9-fluorenylmethyloxycarbonylDMF: dimethylformamide

HOBT: N-hydroxybenzotriazole

DIPCI: diisopropylcarbodiimide

Example 1 Synthesis of a Peptide of the Formula (4)

A peptide (1.5 g) prepared in Preparation 1 as described below andBoc-Cys(Npys)-OH (600 mg) were mixed in dimethylsulfoxide (20 ml) andthe mixture was stirred at room temperature for 20 minutes. Acetonitrile(600 ml) was added to the reaction mixture, and the mixture was stirredon ice, and the resulting precipitate was collected by filtration. Thesolid on the filter was washed with acetonitrile and diethyl etherfollowed by drying under reduced pressure to prepare the peptide (1.65g) in which Boc-Cys-OH was bounded by disulfide bonding. The resultingpeptide (0.53 g) was dissolved in trifluoroacetic acid (5 ml) and thesolution was stirred at room temperature for 10 minutes. Aftertrifluoroacetic acid was evaporated under reduced pressure, the residuewas dissolved in a mixed solution of acetonitrile-acetic acid-water(10/10/90) (110 ml) followed by purification using HPLC.

Pump: LC-8A type (SHIMADZU CORPORATION)Column: YMC ODS-A 3 cmΦ×25 cmL, 10 μm

Eluent 1: H₂O/0.1% TFA Eluent 2: CH₃CN/0.1% TFA

Flow rate: 20 ml/min

Detection: UV220 nm

The crude peptide solution was charged onto the column equilibrated with5% of Eluent 2. Then, 5% of Eluent 2 was run for 10 minutes and 15% ofEluent 2 was run for 15 minutes and thereafter, the concentration ofEluent 2 was increased by 0.1%/min. Fractions comprising the desiredproduct were collected and acetonitrile was evaporated under reducedpressure followed by lyophilization to prepare the desired peptide (300mg).

Mass spectrometry: LC-ESI/MS m/z=1292 [M+1]⁺ (theoretical value=1291.5)

Example 2 Synthesis of a Peptide of the Formula (5)

The desired peptide (104 mg) was prepared by reaction of a peptide (500mg) prepared in Preparation 2 as described below with Boc-Cys(Npys)-OH(200 mg) followed by removal of Boc group in trifluoroacetic acid andpurification by means of HPLC in the similar way to Example 1.

Mass spectrometry: LC-ESI/MS m/z=1260 [M+1]⁺ (theoretical value=1259.5)

Example 3 Synthesis of a Peptide of the Formula (8)

A peptide (240 mg) prepared in Preparation 1 and2,2′-dithiobis(5-nitropyridine) (60 mg) were mixed in dimethylsulfoxide(6 ml) and the mixture was stirred at room temperature for 1 hour.Reduced glutathione (120 mg) was added to the reaction mixture followedby stirring at 30° C. for 1 hour. After reduced glutathione (60 mg) anddimethylsulfoxide (4 ml) were further added to the reaction mixturefollowed by stirring for 30 minutes, acetonitrile (200 ml) was added tothe reaction mixture and then, the resulting precipitate was collectedby filtration and dried under reduced pressure to prepare a crudepeptide (440 mg). The resulting crude peptide was dissolved in themixture (110 ml) of acetonitrile-acetic-acid and water (10/10/90)followed by purification using HPLC.

Pump: LC-8A type (SHIMADZU CORPORATION)Column: YMC ODS-A 3 cmΦ×25 cmL, 10 μm

Eluent 1: H₂O/0.1% TFA Eluent 2: CH₃CN/0.1% TFA

Flow rate: 20 ml/min

Detection: UV220 nm

The crude peptide solution was charged onto the column equilibrated with10% of Eluent 2. Then, 10% of Eluent 2 was run for 10 minutes and 17% ofEluent 2 was run for 15 minutes and thereafter, the concentration ofEluent 2 was increased by 0.05%/min. Fractions comprising the desiredproduct were collected and acetonitrile was evaporated under reducedpressure followed by lyophilization to prepare the desired peptide (107mg).

Mass spectrometry: LC-ESI/MS m/z=1477 [M+1]⁺ (theoretical value=1477.5)

Example 4 Synthesis of a Peptide of the Formula (9)

A peptide (120 mg) prepared in Preparation 2 and2,2′-dithiobis(5-nitropyridine) (30 mg) were mixed in dimethylsulfoxide(3 ml) and the mixture was stirred at room temperature for 1 hour.Reduced glutathione (30 mg) was added to the reaction mixture followedby stirring at room temperature for 30 minutes and then, water (1 ml)and reduced glutathione (30 mg) were further added to the reactionmixture followed by stirring for 20 minutes. After acetonitrile (100 ml)was added to the reaction mixture, the resulting precipitate wascollected by filtration and dried under reduced pressure to prepare acrude peptide (160 mg). The resulting crude peptide was dissolved in themixture (55 ml) of acetonitrile-acetic acid-water (5/5/45) followed bypurification using HPLC.

Pump: LC-6A type (SHIMADZU CORPORATION)

Column: YMC ODS-A 2 cmΦ×25 cmL, 10 μm

Eluent 1: H₂O/0.1% TFA Eluent 2: CH₃CN/0.1% TFA

Flow rate: 10 ml/min

Detection: UV220 nm

The crude peptide solution was charged onto the column equilibrated with10% of Eluent 2. Then, 10% of Eluent 2 was run for 10 minutes and 17% ofEluent 2 was run for 15 minutes and thereafter, the concentration ofEluent 2 was increased by 0.05%/min. Fractions comprising the desiredproduct were collected and acetonitrile was evaporated under reducedpressure followed by lyophilization to prepare the desired peptide (18mg).

Mass spectrometry: LC-ESI/MS m/z=1445 [M+1]⁺ (theoretical value=1445.5)

Example 5 Synthesis of a Peptide of the Formula (6)

A peptide (240 mg) prepared in Preparation 1 and2,2′-dithiobis(5-nitropyridine) (60 mg) were mixed in dimethylsulfoxide(6 ml) and the mixture was stirred at room temperature for 1 hour.Sodium thioglycolate (100 mg) was added to the reaction mixture followedby stirring at 30° C. for 30 minutes and then, sodium thioglycolate (50mg), dimethylsulfoxide (4 ml) and water (2 ml) were further added to thereaction mixture followed by stirring for 30 minutes. After acetonitrile(200 ml) was added to the reaction mixture, the resulting precipitatewas collected by filtration and dried under reduced pressure to preparea crude peptide (305 mg). The resulting crude peptide was dissolved inthe mixture (330 ml) of acetonitrile-acetic acid-water (30/30/270)followed by filtration and then, the resulting filtrate was purifiedusing HPLC.

Pump: LC-8A type (SHIMADZU CORPORATION)Column: YMC ODS-A 3 cmΦ×25 cmL, 10 μm

Eluent 1: H₂O/0.1% TFA Eluent 2: CH₃CN/0.1% TFA

Flow rate: 20 ml/min

Detection: UV220 nm

The crude peptide solution was charged onto the column equilibrated with10% of Eluent 2. Then, 10% of Eluent 2 was run for 10 minutes and 20% ofEluent 2 was run for 15 minutes and 23% of Eluent 2 was run for 15minutes and thereafter, the concentration of Eluent 2 was increased by0.05%/min. Fractions comprising the desired product were collected andacetonitrile was evaporated under reduced pressure followed bylyophilization to prepare the desired peptide (15 mg).

Mass spectrometry: LC-ESI/MS m/z=1263 [M+1]⁺ (theoretical value=1262.5)

Example 6 Synthesis of a Peptide of the Formula (7)

A peptide (240 mg) prepared in Preparation 2 and2,2′-dithiobis(5-nitropyridine) (60 mg) were mixed in dimethylsulfoxide(6 ml) and the mixture was stirred at room temperature for 1 hour.Sodium thioglycolate (50 mg) was added to the reaction mixture followedby stirring at 30° C. for 30 minutes. In addition, after sodiumthioglycolate (50 mg) was further added to the reaction followed bystirring at 30° C. for 1 hour, acetonitrile (200 ml) was added to thereaction mixture. The resulting precipitate was collected by filtrationand dried under reduced pressure to prepare a crude peptide (194 mg).The resulting crude peptide was dissolved in the mixture (120 ml) ofacetonitrile-acetic acid-water (10/20/90) followed by filtration andthen, the resulting filtrate was purified using HPLC.

Pump: LC-8A type (SHIMADZU CORPORATION)Column: YMC ODS-A 3 cmΦ×25 cmL, 10 μm

Eluent 1: H₂O/0.1% TFA Eluent 2: CH₃CN/0.1% TFA

Flow rate: 20 ml/min

Detection: UV220 nm

The crude peptide solution was charged onto the column equilibrated with10% of Eluent 2. Then, 10% of Eluent 2 was run for 10 minutes and 18% ofEluent 2 was run for 15 minutes and thereafter, the concentration ofEluent 2 was increased by 0.1%/min. Fractions comprising the desiredproduct were collected and acetonitrile was evaporated under reducedpressure followed by lyophilization to prepare the desired peptide (30mg).

Mass spectrometry: LC-ESI/MS m/z=1230 [M+1]⁺ (theoretical value=1230.4)

Example 7 Synthesis of a Peptide of the Formula (4) 1. Synthesis ofProtected Peptide Resin(Boc-Cys(Boc-Cys-OH)-Tyr(tBu)-Thr(tBu)-Trp(Boc)-Asn(Trt)-Gln(Trt)-Met-Asn(Trt)-Leu-Alko-Resin)

Fmoc-Leu-Alko-resin (wherein Alko is p-alkoxybenzyl alcohol) (10 g)(0.74 mmol/g, Watanabe Chemical Industries, Ltd.) was charged in areaction vessel (500 ml, Type ACT90 solid phase synthesizer, AdvancedChemTech) and washed once with DMF or the like (Process 1). The resinwas then treated with 25% piperidine (3 minutes×1, and 15 minutes×1) tocleave the Fmoc group (Process 2), and washed again with DMF or the like(Process 6) to remove piperidin. To the reaction vessel was added asolution of Fmoc-Asn(Trt)-OH (13.25 g) and HOBT (1-hydroxybenzotriazole)(3.4 g) in NMP (N-methylpyrrolidinone) (200 ml). After adding DIPCI(N,N′-diisopropylcarbodiimide) (3.42 ml), the mixture was stirred atroom temperature for 60 minutes (Process 3). Then, the resin was washedwith NMP (Process 4) and coupling reaction was conducted again usingFmoc-Asn(Trt)-OH (13.25 g), HOBT (3.4 g) and DIPCI (3.42 ml) (Process3). After the resin was washed (Process 6), the resin was stirred in 25%Ac₂O (acetic anhydride) for 3 minutes×1 and for 15 minutes×2 to cap theunreacted amino groups (Process 5). The resin was washed (Process 6)followed by deprotection (Process 2) and washing (Process 6) to prepareH-Asn(Trt)-Leu-Alko-resin. A coupling reaction was conducted usingFmoc-Met-OH (8.25 g), Fmoc-Gln(Trt)-OH (13.56 g), Fmoc-Asn(Trt)-OH(13.25 g), Fmoc-Trp(Boc)-OH (11.69 g), Fmoc-Thr(tBu)-OH (8.82 g),Fmoc-Tyr(tBu)-OH (10.2 g), and (Boc-Cys-OH)₂ (19.56 g) in a similar way,with the proviso that coupling was repeated three times in the case ofdifficulty in the coupling. After (Boc-Cys-OH)₂(N,N′-t-butoxycarbonylcystine) located at N-terminal was condensed,washing (Process 6) was conducted followed by washing with diethylether(200 ml) twice and drying under reduced pressure to prepareBoc-Cys(Boc-Cys-OH)-Tyr(tBu)-Thr(tBu)-Trp(Boc)-Asn(Trt)-Gln(Trt)-Met-Asn(Trt)-Leu-Alko-Resin(the peptide-resin of the formula (10)) (22.87 g). The above processesfor synthesis are summarized in the following Table.

TABLE 1 <Processes for Synthesis> Number of Process Reagent treatmentTime(min) 1) Washing DMF 200 ml × 3 0.3 MeOH 200 ml × 1 0.3 DMF 200 ml ×3 0.3 2) Deprotection 25% 200 ml 3.0 piperidine/ DMF 200 ml 15.0 3)Coupling Amino-protected amino acid (3 eq. for each) 60 × 1 HOBT (3 eq.)DIPCI (3 eq.)/NMP 200 ml 4) Washing NMP 200 ml × 2 0.3 5) Capping 25%acetic 200 ml 3.0 anhydride/ DMF 200 ml 15.0 6) Washing DMF 200 ml × 50.3 MeOH 200 ml × 1 0.3 DMF 200 ml × 5 0.3

2. Deprotection of Protected Peptide Resin

The mixture (200 ml) of trifluoroaceticacid/ethanediol/H₂O/triisopropylsilane (94/2.5/2.5/1) was added to theprotected peptide resin(Boc-Cys(Boc-Cys-OH)-Tyr(tBu)-Thr(tBu)-Trp(Boc)-Asn(Trt)-Gln(Trt)-Met-Asn(Trt)-Leu-Alko-Resin (22.87 g) obtained in accordance withthe processes above, and the mixture was stirred at room temperature for4 hours. After the reaction product was filtrated, the filtrate wasadded to diethylether (400 ml) with cooling on ice. The resultingprecipitate was collected by filtration using glass filter and then,washed with diethylether and dried under reduced pressure to prepare thecrude peptide (8.27 g).

3. Purification of Crude Peptide

The resulting crude peptide (2.76 g) was dissolved in the mixed solutionof 20% acetic acid aqueous solution (1400 ml) and acetonitrile (35 ml)and the resulting insoluble substances were removed by filtration. Theresulting crude peptide solution was purified using reversed-phaseliquid chromatography.

Pump: LC-8A type (SHIMADZU CORPORATION)Column: YMC ODS-A 5 cmΦ×50 cmL, 15-30 μm

Eluent 1: H₂O/0.1% TFA Eluent 2: CH₃CN/0.1% TFA

Flow rate: 60 ml/min

Detection: UV280 nm

The crude peptide solution was charged onto the column equilibrated with10% of Eluent 2. Then, 10% of Eluent 2 was run for 30 minutes andthereafter, the concentration of Eluent 2 was increased to 34% over 120minutes. Fractions comprising the desired product were collected andacetonitrile was evaporated under reduced pressure followed bylyophilization to prepare the desired peptide:H-Cys(H-Cys-OH)-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH (the peptide of theformula (4)) (0.91 g).

Test Example 1 Immunization of a Mouse (1)

An immunogenicity of each antigen peptide prepared by Examples 1-6 wasevaluated using HLA-A2402/K^(b) transgenic mice (see WO 02/47474 andhereinafter, the mice may be referred to as HLA-A24 mice). Threetransgenic mice were used in the immunization of each peptide toevaluate the immunogenicity.

A pharmaceutical composition comprising each synthetic peptide wasprepared as follows. Each of the synthetic peptides of Examples 1-3,5,6was adjusted to 40 mg/ml in DMSO. Then, the solution (32.5 μl) was mixedwith water for injection (540 μl). Further, the resulting solution (550μl) was mixed with Freund's incomplete adjuvant (700 μl) (MontanideISA51) using a glass syringe to prepare water-in-oil emulsion. Thepeptide of Example 4 was adjusted to 50 mg/ml in DMSO and synthetichelper paptide (FNNFTVSFWLRVPKVSASHLE, SEQ ID No:5) was also adjusted to20 mg/ml in DMSO. Then, 30 μl of both peptide solutions were mixed inwater for injection (540 μl) followed by being mixed with the equalamount of Freund's incomplete adjuvant (IFA) to prepare water-in-oilemulsion.

The resulting preparation (200 μl) was injected into an HLA-A2402/K^(b)transgenic mouse intradermally in the base of the tail for immunization.7-8 days after initiation of the experiment, spleen was removed andgrounded on the frosted part of glass slide, and splenocytes werecollected and prepared. A portion of the splenocytes undergone hemolysistreatment with ACK buffer (0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH7.2-7.4) was pulsed with the antigen peptide used in the immunization(100 μg/ml) for 1 hour and seeded into 24-well plate (7×10⁶ cells/well).Simultaneously, splenocytes not pulsed with any peptide (7×10⁵cells/well) were added together, and stimulated in vitro and cultured at37° C. for 5-6 days. The in vitro stimulation was carried out inRPMI-1640 medium supplemented with 10% FCS, 10 mM HEPES, 20 mML-glutamine, 1 mM sodium pyruvate, 1 mM MEM nonessential amino acids, 1%MEM vitamin and 55 μM 2-mercaptoethanol.

5-6 days after initiation of restimulation, the test for cytotoxicactivity was conducted according to the conventional manner.EL4-A2402/K^(b) cells obtained by transforming EL-4 cells (DAINIPPONPHARMACEUTICAL CO., LTD., Catalogue No. 06-039) with an expressionvector encoding HLA-A2402/K^(b), and the EL4-A2402/K^(b) cells pulsedwith antigen peptide: WT1₂₃₅₋₂₄₃ or WT1₂₃₅₋₂₄₃ (2M→Y) were used astarget cells (T). Specifically, cells pulsed with WT1₂₃₅₋₂₄₃ (2M→Y) wereused for evaluation of peptides of Examples 1, 3 and 5, and cells pulsedwith WT1₂₃₅₋₂₄₃ were used for evaluation of peptides of Examples 2, 4and 6. These cells were labeled with ⁵¹Cr (1.85 MBq/10⁶ cells) andpulsed with the peptide at 100 μg/ml for an hour (The labeling wascarried out for 2 hours, and 1 hour after the initiation of labeling,the peptide was added). ⁵¹Cr release assay (J. Immunol., 159:4753, 1997)was conducted to determine the cytotoxic activity of in vitro culturedsplenocytes (E) to target cells (T). In this assay, E/T ratio was 80.

The results are shown in FIGS. 1-7. FIGS. 1-6 correspond to cytotoxicactivities of peptide compounds of Examples 1-6 respectively. In thefigures, the vertical axis shows cytotoxic activity (Specific Lysis),and the horizontal axis shows results of each of three miceindividually. In addition, the results of dose dependence of thecompound of Example 1 are shown in FIG. 7. In the figure, the verticalaxis shows cytotoxic activity (Specific Lysis), and the horizontal axisshows individually administered dose (600 μg, 200 μg, 60 μg and 20 μg).In the figure, three mice were used per each dose, and mean and standarddeviation (S.D.) of the cytotoxic activities are shown. As clearlyunderstood from the figures, it was found that all of the syntheticpeptides have CTL induction activity, that is to say, immunogenicity.

Test Example 2 Immunization of a Mouse (2)

The peptide of Example 1 was adjusted to 40 mg/ml in DMSO. Then, thesolution (32.5 μl) was mixed with water for injection (540 μl). Further,the resulting solution (550 μl) was mixed with Freund's incompleteadjuvant (700 μl) (Montanide ISA51 (registered trademark)) (SEPPIC, Inc,Paris, France) using a glass syringe to prepare water-in-oil emulsion.

The resulting preparation (200 μl) was injected into an HLA-A2402/K^(b)transgenic mouse intradermally in the base of the tail for immunization.7 days after initiation of the experiment, spleen was removed andsplenocytes were prepared in a conventional manner (WO 02/47474). Then,tests were conducted using mouse IFN gamma ELISPOT set (Enzyme-LinkedImmunospot) (Fujisawa, catalog No. BD-551083). The test was conductedaccording to the instructions attached to the set. 5×10⁵ cells/well ofsplenocytes were plated and culture medium containing the peptide ofExample 1, wild type peptide (WT1₂₃₅₋₂₄₃) or a peptide derived frominfluenza virus (ASNENMETM, negative control peptide which does not bindto HLA-A24) was added at the final concentration of 10⁻⁶M followed byincubation using CO₂ incubator at 37° C. for 18 hours.

According to the attached instructions, the plate was washed and thenumber of spots was detected using KS Elispot Research System (CarlZeiss). Elispot method is known as an alternative to a test forcytotoxic activity (J. Immunological Methods, 1995, 181, 45-54). Theresults are shown in FIG. 8. As a result, it was found that the peptideof Example 1 could induce HLA-A24 specific cell-mediated immunity whichcross reacted with the wild type peptide.

Test Example 3 Test Using Human PBMC

Peripheral blood (50 ml) was taken using heparinized vacuum bloodcollection tube from HLA-A24 positive healthy subjects. The blooddiluted two fold with PBS (−) was overlayered on Ficoll-Paque (AmershamBiosciences), the amount of which was half of the amount of the blood,followed by centrifugation for 20 minutes at 2000 rpm. A cell layercontaining the peripheral blood mononuclear cells (PBMC) was collectedand 3-4 times the amount of PBS (−) was added thereto, followed bycentrifugation for 10 minutes at 1800 rpm. The cell pellet was washedtwice with PBS (−) and PMBC was collected.

The PMBC was suspended in medium for culturing lymphocyte (RPMI1640: AIMV=1:1, NEAA, 10% FCS) and cultured in a culture flask for two hours andnonadherent cells were collected. Using CD8⁺ T cell isolation kit II(Miltenyi Biotec), CD8 positive T cells were collected among thenonadherent cells. The adherent cells were cultured in the medium forculturing lymphocyte containing GM-CSF (1000 U/ml) and IL-4 (1000 U/ml)for 7 days. The floating cells were collected as an antigen-presentingcell fraction containing dendritic cells (DC). The collected cells werefrozen for preservation until they are used for experiments.

The DC fraction prepared as above was pulsed with the peptide of Example1 (50 μg/ml) by incubating the cells with the peptide in AIM-V mediumcontaining mitomycin (50 μg/ml) for one hour. After washing with themedium three times, the peptide of Example 1 was further added (50μg/ml) for one hour pulse to prepare antigen-presenting cells. On Day 0,antigen-presenting cells pulsed with the peptide were added to CD8positive T cells to conduct the first stimulation, and the cell culturewas started in medium for culturing lymphocyte containing IL-7 (10ng/ml) using 24-well plate. On Day 7, T cells were collected and afterwashing them, peptide stimulation using antigen-presenting cells pulsedwith the peptide was conducted in a similar way to the firststimulation. On the next day (Day 8), IL-2 was added so that theconcentration was 50 U/ml. On Day 14, T cells were collected and thethird stimulation was conducted in a similar way to the first or secondstimulation. On the next day (Day 15), IL-2 was added so that theconcentration was 50 U/ml. On Day 21, T cells were collected and frozenfor preservation.

1×10⁵ cells of T cells of Day 21 were added to 2×10⁴ cells of HLA-A2402expressing VA13/A2402 cells pulsed with or without each of the peptides(wild type peptide (WT1₂₃₅₋₂₄₃), modified peptide (WT1₂₃₅₋₂₄₃(2M→Y)) orthe peptide of Example 1) and 18 hours later, the supernatant wascollected to determine the amount of IFN-γ using ELISA.

The results of the peptide specific reactivity of T cells stimulatedwith the synthetic peptide of Example 1 were shown in FIG. 9. T cellsstimulated with the synthetic peptide of Example 1 did not react withcells pulsed without the peptide, but they sufficiently reacted withcells pulsed with the peptide of Example 1. Thus, it was found that Tcells specific for the peptide were induced. In addition, the induced Tcells specific for the peptide reacted with the cells pulsed with thewild type peptide (WT1₂₃₅₋₂₄₃) as well as the cells pulsed with themodified peptide (WT1₂₃₅₋₂₄₃ (2M→Y)).

Preparation 1 1. Synthesis of Protected Peptide Resin(H-Cys(Trt)-Tyr(tBu)-Thr(tBu)-Trp(Boc)-Asn(Trt)-Gln(Trt)-Met-Asn(Trt)-Leu-Alko-Resin)

Fmoc-Leu-Alko-resin (wherein Alko is p-alkoxybenzyl alcohol) (10 g)(0.82 mmol/g, Watanabe Chemical Industries, Ltd.) was charged in areaction vessel (500 ml, Type ACT90 solid phase synthesizer, AdvancedChemTech) and washed once with DMF or the like (Process 1). The resinwas then treated with 25% Pip (piperidine) (3 minutes×1, and 15minutes×1) to cleave the Fmoc group (Process 2), and washed again withDMF or the like (Process 1) to remove Pip. To the reaction vessel wasadded a solution of Fmoc-Asn(Trt)-OH (24.46 g) and HOBT(1-hydroxybenzotriazole) (6.28 g) in NMP (N-methylpyrrolidinone) (200ml). After adding DIPCI (N,N′-diisopropylcarbodiimide) (6.3 ml), themixture was stirred at room temperature for 30 minutes (Process 3).Thirty minutes later, the resin was washed with NMP (Process 4), andsubjected to the coupling reaction once again using Fmoc-Asn(Trt)-OH(24.46 g), HOBT (6.28 g) and DIPCI (6.3 ml) (Process 5) to synthesizeFmoc-Asn(Trt)-Leu-Alko resin. The resultant resin was then converted toH-Asn(Trt)-Leu-Alko-resin by the deprotection of Process 2. Afterwashing (Process 1), Fmoc-Met-OH 15.23 g, Fmoc-Gln(Trt)-OH 25.04 g,Fmoc-Asn(Trt)-OH 24.46 g, Fmoc-Trp(Boc)-OH 21.59 g, Fmoc-Thr(tBu)-OH16.3 g, Fmoc-Tyr(tBu)-OH 18.84 g and Fmoc-Cys(Trt)-OH 24.01 g were addedin series to conduct the coupling reaction (Process 3). In the couplingreaction using Fmoc-Thr(tBu)-OH, the reaction was repeated three times,and the resulting resin was washed with DMF and treated with 25% A_(C2)O(acetic anhydride) (15 minutes×2) for the capping of unreacted aminogroups. After condensation of the N-terminal Fmoc-Cys(Trt)-OH, thedeprotection (Process 2) and washing (Process 6) were conducted toobtainH-Cys(Trt)-Tyr(tBu)-Thr(tBu)-Trp(Boc)-Asn(Trt)-Gln(Trt)-Met-Asn(Trt)-Leu-Alko-Resin.The above processes for synthesis are summarized in the following Table.

TABLE 2 <Processes for Synthesis> Number of Process Reagent treatmentTime(min) 1) Washing DMF 200 ml × 6  0.3 MeOH 200 ml × 1  0.3 DMF 200 ml× 3  0.3 2) Deprotection 25% 200 ml  3.0 piperidine/ DMF 200 ml 15.0 3)Coupling Amino-protected amino acid (5 eq. for each) 30 × 1 HOBT (5 eq.)DIPCI (5 eq.)/NMP 200 ml 4) Washing NMP 200 m1 × 2  0.3 5) CouplingAmino-protected amino acid (5 eq. for each) 30 × 1 HOBT (5 eq.) DIPCI (5eq.)/NMP 200 ml 6) Washing DMF 200 ml × 5  0.3 MeOH 200 ml × 1  0.3 DMF200 ml × 2  0.3

2. Deprotection of Protected Peptide Resin

The mixed solution of trifluoroaceticacid/ethanediol/H₂O/triisopropylsilane (94/2.5/2.5/1) (100 ml) was addedto the protected peptide resin(H-Cys(Trt)-Tyr(tBu)-Thr(tBu)-Trp(Boc)-Asn(Trt)-Gln(Trt)-Met-Asn(Trt)-Leu-Alko-Resin)(10.0 g) as prepared above and the resulting mixture was stirred at roomtemperature for 4 hours. The reaction mixture was added tot-butylmethylether (625 ml) with cooling on ice and the resultingmixture was stirred for 15 minutes followed by filtration using a glassfilter to obtain insoluble substances. After the residue on the filterwas washed with t-butylmethylether (about 100 ml) five times, theresidue on the filter was extracted with 6M guanidine hydrochlorideaqueous solution (1 L) to prepare the crude peptide solution.

3. Purification of Crude Peptide

The resulting crude peptide solution was purified using reversed-phaseliquid chromatography.

Pump: LC-8A type (SHIMADZU CORPORATION)Column: YMC ODS-A 5 cmΦ×50 cmL, 15-30 μm

Eluent 1: H₂O/0.1% TFA Eluent 2: CH₃CN/0.1% TFA

Flow rate: 60 ml/min

Detection: UV220 nm

The crude peptide solution was charged onto the column which wasequilibrated with 10% of Eluent 2 and kept in a water bath at 50° C.After 10% of Eluent 2 was run for 30 minutes, the concentration ofEluent 2 was increased to 20% over 40 minutes and further increased to40% over 360 minutes. Fractions comprising a desired product werecollected and acetonitrile was evaporated under reduced pressurefollowed by lyophilization to prepare the desired peptide:H-Cys-Tyr-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH (SEQ ID No:2) (1.50 g).

amino acid analysis

Hydrolysis: 1% phenol/6N aqueous hydrochloric acid, 110° C., 10 hours

Analysis method: ninhydrin method

Asx:1.96(2) Thr:1.05(1) Glx:1.06(1) Met:1.05(1) *Leu:(1) Tyr:0.87(1)

*) Leu=standard amino acid Theoretical value is described in ( ).

Mass analysis: LC-ESI/MS m/z=1173 [M+1]⁺ (theoretical value=1172.5)

Amino acid sequence analysis: The amino acid residues from Tyr atposition 2 from the N-terminal to Leu at the C-terminal weresequentially confirmed.

Preparation 2 1. Synthesis of Protected Peptide Resin(H-Cys(Trt)-Met-Thr(tBu)-Trp(Boc)-Asn(Trt)-Gln(Trt)-Met-Asn(Trt)-Leu-Alko-Resin)

Fmoc-Leu-Alko-resin (wherein Alko is p-alkoxybenzyl alcohol) (10 g)(0.81 mmol/g, Watanabe Chemical Industries, Ltd.) was charged in areaction vessel (500 ml, Type ACT90 solid phase synthesizer, AdvancedChemTech) and washed once with DMF or the like (Process 1). The resinwas then treated with 25% Pip (piperidine) (3 minutes×1, and 15minutes×1) to cleave the Fmoc group (Process 2), and washed again withDMF or the like (Process 1) to remove Pip. To the reaction vessel wasadded a solution of Fmoc-Asn(Trt)-OH (24.17 g) and HOBT(1-hydroxybenzotriazole) (6.2 g) in NMP (N-methylpyrrolidinone) (200ml). After adding DIPCI (N,N′-diisopropylcarbodiimide) (6.2 ml), themixture was stirred at room temperature for 30 minutes (Process 3).Thirty minutes later, the resin was washed with NMP (Process 4), andsubjected to the coupling reaction once again using Fmoc-Asn(Trt)-OH(24.17 g), HOBT (6.2 g) and DIPCI (6.2 ml) (Process 5) to synthesizeFmoc-Asn(Trt)-Leu-Alko resin. The resultant resin was then converted toH-Asn(Trt)-Leu-Alko-resin by the deprotection of Process 2. Afterwashing (Process 1), Fmoc-Met-OH 15.05 g, Fmoc-Gln(Trt)-OH 24.73 g,Fmoc-Asn(Trt)-OH 24.17 g, Fmoc-Trp(Boc)-OH 21.33 g, Fmoc-Thr(tBu)-OH16.1 g, Fmoc-Met-OH 15.05 g and Fmoc-Cys(Trt)-OH 23.72 g were added inseries to conduct the coupling reaction (Process 3). In the couplingreaction using Fmoc-Thr(tBu)-OH, the reaction was repeated three times,and the resulting resin was washed with DMF and treated with 25% A_(C2)O(acetic anhydride) (15 minutes×2) for the capping of unreacted aminogroups. After condensation of the N-terminal Fmoc-Cys(Trt)-OH, thedeprotection (Process 2) and washing (Process 6) were conducted toobtainH-Cys(Trt)-Met-Thr(tBu)-Trp(Boc)-Asn(Trt)-Gln(Trt)-Met-Asn(Trt)-Leu-Alko-Resin.The above processes for synthesis are the same as described in the tableof Preparation 1.

2. Deprotection of Protected Peptide Resin

The mixture of trifluoroacetic acid/ethanediol/H₂O/triisopropylsilane(94/2.5/2.5/1) (130 ml) was added to the protected peptide resin(H-Cys(Trt)-Met-Thr(tBu)-Trp(Boc)-Asn(Trt)-Gln(Trt)-Met-Asn(Trt)-Leu-Alko-Resin)(13.0 g) as prepared above and the resulting mixture was stirred at roomtemperature for 4 hours. The reaction mixture was added to diethylether(800 ml) with cooling on ice and the resulting mixture was stirred for15 minutes followed by filtration using a glass filter to obtaininsoluble substances. After the residue on the filter was washed withdiethylether (about 100 ml) five times, the residue on the filter wasextracted with 6M guanidine hydrochloride aqueous solution (1.3 L) toprepare the crude peptide solution.

3. Purification of Crude Peptide

The resulting crude peptide solution was purified using reversed-phaseliquid chromatography.

Pump: LC-8A type (SHIMADZU CORPORATION)Column: YMC ODS-A 5 cmΦ×50 cmL, 15-30 μm

Eluent 1: H₂O/0.1% TFA Eluent 2: CH₃CN/0.1% TFA

Flow rate: 60 ml/min

Detection: UV220 nm

The crude peptide solution was charged onto the column which wasequilibrated with 10% of Eluent 2 and kept in a water bath at 50° C.After 10% of Eluent 2 was run for 30 minutes, the concentration ofEluent 2 was increased to 20% over 40 minutes and further increased to40% over 360 minutes. Fractions comprising a desired product werecollected and acetonitrile was evaporated under reduced pressurefollowed by lyophilization to prepare the desired peptide:H-Cys-Met-Thr-Trp-Asn-Gln-Met-Asn-Leu-OH (SEQ ID No:1) (2.32 g).

amino acid analysis

Hydrolysis: 4N methanesulfonic acid, 110° C., 17 hours

Analysis method: ninhydrin method

Asx:1.87(2) Thr:0.93(1) Glx:0.95(1) Met:1.72(2) *Leu:(1) Trp:0.80(1)

*) Leu=standard amino acid Theoretical value is described in ( ).

Mass analysis: LC-ESI/MS m/z=1141 [M+1]⁺ (theoretical value=1140.5)

Amino acid sequence analysis: The amino acid residues from Met atposition 2 from the N-terminal to Leu at the C-terminal weresequentially confirmed.

INDUSTRIAL APPLICABILITY

The peptide compounds of the present invention are useful as an activeingredient comprised in a medicament for cancer immunotherapy.

SEQUENCE LISTING FREE TEXT

SEQ ID No.1: peptide derivativeSEQ ID No.2: peptide derivativeSEQ ID No.3: peptide derivativeSEQ ID No.4: peptide derivativeSEQ ID No.5: helper synthetic peptide

1-16. (canceled)
 17. A method for treating cancer comprising:administering to a subject in need thereof who is positive for HLA-A24and positive for WT1 a therapeutically effective amount of a compound offormula (1) or a pharmaceutically acceptable salt thereof; whereinformula (1) is:

wherein X is a tyrosine residue or a methionine residue; each of Y and Zis independently selected from the group consisting of a single bond anda divalent peptide group consisting of 1-10 amino acid residues, R¹ ishydrogen or alkyl, R² is hydroxyl, amino, alkylamino or dialkylamino, R³is hydrogen, alkyl, amino, alkylamino, dialkylamino or substituted orunsubstituted alkylcarbonylamino, R⁴ is hydrogen, alkyl, carboxy,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl or a group of formula (2):

wherein W is an amino acid residue, m is 1 or 2, and n is an integer of0-2, with the proviso that when n is 0, then R³ is hydrogen or alkyl.18. The method of claim 17 that comprises administering a compound ofpharmaceutically acceptable salt of a compound of formula (1) whereineach of R1 and R2 is hydrogen.
 19. The method of claim 17 that comprisesadministering a compound of pharmaceutically acceptable salt of acompound of formula (1), wherein R3 is alkylcarbonylamino or substitutedalkylcarbonylamino.
 20. The method of claim 17 that comprisesadministering a compound of pharmaceutically acceptable salt of acompound of formula (1) that is:


21. The method of claim 17 that comprises administering a compound ofpharmaceutically acceptable salt of a compound of formula (1) that is:


22. The method of claim 17, wherein said compound of formula (1) isadministered on an antigen-presenting cell that comprises a complexbetween HLA-A24 antigen and the compound of formula (1) or apharmaceutically acceptable salt thereof.
 23. The method of claim 17,wherein the cancer expresses WT1.
 24. The method of claim 17, whereinthe cancer is selected from the group consisting of leukemia,myelodysplastic syndrome, multiple myeloma, malignant lymphoma, gastriccancer, colon cancer, lung cancer, breast cancer, embryonal cancer,hepatic cancer, skin cancer, bladder cancer, prostate cancer, uterinecancer, cervical cancer, and ovarian cancer.
 25. A method for treatingcancer comprising administering to a subject in need thereof a CTLinduced by a compound of formula (1) or a pharmaceutically acceptablesalt thereof; wherein formula (1) is:

wherein X is a tyrosine residue or a methionine residue; each of Y and Zis independently selected from the group consisting of a single bond anda divalent peptide group consisting of 1-10 amino acid residues, R¹ ishydrogen or alkyl, R² is hydroxyl, amino, alkylamino or dialkylamino, R³is hydrogen, alkyl, amino, alkylamino, dialkylamino or substituted orunsubstituted alkylcarbonylamino, R⁴ is hydrogen, alkyl, carboxy,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl or a group of formula (2):

wherein W is an amino acid residue, m is 1 or 2, and n is an integer of0-2, with the proviso that when n is 0, then R³ is hydrogen or alkyl.26. The method of claim 25, wherein said CTL recognizes a complex ofHLA-A24 antigen and the compound of formula (1) or a pharmaceuticallyacceptable salt thereof.
 27. The method of claim 25, wherein the CTL isautologous to the subject.